The expression, structure, and function of VLAs (beta1 integrins) on mouse thymocytes were analyzed, as a first step toward understanding their role in T cell development. Two major forms of beta1 were detected, which differed in their extent of N-glycosylation and sialylation. The larger (135 kDa, nonreduced) more basic beta1 was the only form on nonmature (J11d+) adult thymocytes, being expressed on fetal thymocytes from day 14 of gestation through birth. The smaller (120 kDa, non reduced), more acidic beta-chain was found exclusively on mature (J11d-) thymocytes and peripheral lymphocytes. VLA alpha-chain expression was also analyzed. Virtually all thymocytes were VLA-alpha4+, -alpha6+. VLA-alpha5 was detected on both J11d+ and J11d- thymocytes by immunoprecipitation. VLA- alpha1 and -alpha2 were immunoprecipitated only from cells within the J11d- population. Only J11d- thymocytes (12 to 15%) bound to fibronectin (via VLA-4 and VLA-5) and binding to laminin (via VLA-6) was two to fourfold higher in J11d+ compared to J11d- thymocytes (60 to 80% vs 20 to 30%). The high percentage of J11d+ thymocytes adhering to laminin suggest that VLA-6 exists in an activated state on most thymocytes, in contrast to resting peripheral T cells. These data show that major changes in VLA glycosylation, expression, and extracellular matrix binding (ECM) occur as thymocytes mature, supporting the hypothesis that VLA-ECM interactions play a role in T cell ontogeny. We have engineered soluble chimeric TCR/IgG, containing the extracellular domains of the mouse Vgamma1.1-Cgamma4 and Vdelta6.2-Cdelta (V, variable; C, constant) TCR chains fused to the hinge region CH2 (H, heavy) and CH3 domains of human IgG1 heavy chain, and expressed them by transient transfection in COS cells. To gain insight into the developmentally regulated expression of the mouse TCR Vdelta-gene segments, we have investigated the role of 5' promoter region of the Vdelta1-gene. In vivo genomic footprinting localized several protein-DNA interactions to the stretch of DNA shown to have transcriptional activity. A computer analysis revealed that segments of DNA participating in these protein-DNA interactions were identical to the previously described cyclic AMP response element (CRE), E box, and leukemia virus E26 cis-acting elements. Transient transfection assays revealed that the CRE, E box, and Ets elements work together in driving promoter activity and that the CRE and Ets elements are the most important for driving transcription. These data indicate that multiple transcription factors acting in concert are responsible for Vdelta1 gene expression.